Alphaviruses and flaviviruses include important human pathogens such as the encephalitic alphaviruses and chikungunya virus, and the flaviviruses dengue, yellow fever, and West Nile virus. Many of these viruses are classified as category A, B or C priority pathogens, but in spite of their medical importance there are no available antiviral therapies. Alphaviruses and flaviviruses infect cells by a membrane fusion reaction that is triggered by low pH during virus endocytic uptake and mediated by structurally similar membrane fusion proteins termed class II. These fusion proteins are synthesized together with a companion protein that dimerizes with the fusion protein and protects it from low pH during transit to the plasma membrane. Late in the secretory pathway the companion proteins are proteolytically processed by cellular furin, thereby priming the dimer to dissociate a low pH during virus entry. The fusion proteins then drive virus membrane fusion by inserting a hydrophobic fusion loop into the target membrane and refolding to a hairpin-like homotrimer. This proposal will address the following critical questions in the mechanism and biogenesis of class II fusion proteins: 1. The roles of E3 and E2 interactions during alphavirus entry and biogenesis. Furin processing converts the alphavirus p62 companion protein to mature E2 and an E3 peptide. We will define the critical dimer dissociation steps that mediate the initial uncovering of the fusion loop on E1 and the subsequent complete dissociation of the dimer to permit E1 homotrimer formation. We will determine the function of E3 in protecting E1 from low pH during virus biogenesis, and the role of a conserved E2 CXXC motif in promoting E1 disulfide bond formation. 2. Molecular mechanisms of class II protein-membrane interaction. We will use innovative fluorescence methods to study the mechanism of membrane insertion of the alphavirus fusion protein, and we will determine the specific protein loops involved, the properties of insertion and the role of cholesterol in this process. We will develop a novel cross-linking assay to define the cooperative lateral interactions between E1 trimers in the target membrane and we will determine their role in virus-membrane fusion. 3. Novel class II pH protection mechanisms. Recent studies demonstrate that the rubella virus (RuV) fusion protein E1 has a typical class II structure, but its companion protein E2 is not proteolytically processed. RuV E1 is thus an important paradigm for a growing group of low pH-triggered viral class II proteins in which fusion is not regulated by companion protein processing. We will define the novel mechanisms that protect the RuV fusion protein from low pH inactivation during virus biogenesis.